Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis. Cold-acclimated G. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis. Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo, with implications for choosing new molecules for cryopreservation.

• Keywords
• Gryllus veletis, acclimation, cold tolerance, cryopreservation, cryoprotectants, freeze tolerance,
• MeSH
• Acclimatization * MeSH
• Longevity MeSH
• Gryllidae growth & development   physiology   MeSH
• Hemolymph physiology   MeSH
• Cryoprotective Agents metabolism   MeSH
• Metabolomics MeSH
• Cold Temperature * MeSH
• Nymph growth & development   physiology   MeSH
• Proline metabolism   MeSH
• Trehalose metabolism   MeSH
• Fat Body physiology   MeSH
• Freezing MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cryoprotective Agents MeSH
• Proline MeSH
• Trehalose MeSH